Enzymatic synthesis of 20-methylseleno-modified RNA†

Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 20-methylseleno (20-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 20-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 20-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 20methylseleno-20-deoxyribonucleoside triphosphates of cytosine and uridine (20-SeCH3–CTP and 20SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 20-SeCH3–CMP and 20-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 20-SeCH3modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.